Cyclo rgdyk

IGF1 receptor (IGF1R) expression was evaluated fluorometrically by the PE-conjugated anti-Human CD221 (IGF1R; clone1H7; BD Pharmingen) monoclonal antibody. Mouse IgG1 (clone P3.6.2.8.1; Thermo Fisher, Dreieich, Germany) was used as the control isotype. In ongoing experiments, the tumor cells were activated with LONG^®R3IGF-1 (100 ng/mL) and adhesion, chemotaxis, and the integrin α2 (clone 12F1-H6), αV (clone 13C2), and β1 (clone MAR4) surface expression was analyzed. Adhesion to immobilized collagen and chemotaxis was also evaluated when tumor cells were serum starved for 12 h and stimulated with IGF-1 and simultaneously blocked with anti-integrin α2 (clone P1E6) or anti-integrin β1 (clone 6S6), both mouse mAb (all obtained from Merck Millipore, Burlington, MA, USA). BTT3033 (Bio-Techne, MN, USA), a selective inhibitor of the integrins α2β1, or Cyclo(RGDyK) (Selleckchem, TX, USA), targeted against integrin αVβ3, were also employed to determine whether cross-communication between IGF-evoked Akt signaling and integrin expression exists. HepG2 cells were activated with IGF and integrin α2 and β1 expression were measured by flow cytometry and compared to the integrin expression in HepG2 cells treated with the AKT inhibitor MK-2206 (Selleckchem, TX, USA).

Cyclo (RGDyK) (CYC, catalog S7844) was purchased from Selleck chem, USA. Anti-p-ERK (catalog 4377), ERK1/2 (catalog 4695), p-FAK (catalog 3283), FAK (catalog 3285), β-actin (catalog 4970), and HRP-conjugated secondary (catalog 7074) antibodies were purchased from Cell Signaling Tech, USA. DMSO (catalog C6164) and collagenase II (catalog C2-BIOC) were purchased from Sigma, USA.